Stomatal Guard Cells Research Articles

Plastidic protein Cdf1 is essential in Arabidopsis embryogenesis

Arabidopsis cell growth defect factor-1 (Cdf1 in yeast, At5g23040) was originally isolated as a cell growth suppressor of yeast from genetic screening. To investigate the in vivo role of Cdf1 in plants, a T-DNA insertion line was analyzed. A homozygous T-DNA insertion mutant (cdf1/cdf1) was embryo lethal and showed arrested embryogenesis at the globular stage. The Cdf1 protein, when fused with green fluorescent protein, was localized to the plastid in stomatal guard cells and mesophyll cells. A promoter-β-glucuronidase assay found expression of Cdf1 in the early heart stage of embryogenesis, suggesting that Cdf1 was essential for Arabidopsis embryogenesis during the transition of the embryo from the globular to heart stage.

Reconstructing relative genome size of vascular plants through geological time

The strong positive relationship evident between cell and genome size in both animals and plants forms the basis of using the size of stomatal guard cells as a proxy to track changes in plant genome size through geological time. We report for the first time a taxonomic fine-scale investigation into changes in stomatal guard-cell length and use these data to infer changes in genome size through the evolutionary history of land plants. Our data suggest that many of the earliest land plants had exceptionally large genome sizes and that a predicted overall trend of increasing genome size within individual lineages through geological time is not supported. However, maximum genome size steadily increases from the Mississippian (c. 360 million yr ago (Ma)) to the present. We hypothesise that the functional relationship between stomatal size, genome size and atmospheric CO2 may contribute to the dichotomy reported between preferential extinction of neopolyploids and the prevalence of palaeopolyploidy observed in DNA sequence data of extant vascular plants.

Variability of stomata and 45S and 5S rDNAs loci characteristics in two species ofAnthoxanthumgenus:A. aristatumandA. odoratum(Poaceae)

Diploid Anthoxanthum odoratum and tetraploid A. aristatum were compared with respect to stomatal guard cell lengths, and stomatal density at adaxial and abaxial surfaces of the lamina. Further, the genome size of both species was determined by flow cytometry, and the number as well as the chromosomal distribution of 5S and 45S rDNAs were examined using FISH with ribosomal DNA (rDNA) probes. The average length of stomatal guard cells in A. odoratum was shown to be greater than that for A. aristatum, but the ranges overlapped. Moreover, reduction in stomatal frequency was found at higher ploidy levels.The genome size was 6.863 pg/2C DNA for A. aristatum and 13.252 pg/2C DNA for A. odoratum. A. aristatum has four sites of 5S rDNA in its root-tip meristematic cells, whereas A. odoratum has six. Both species have six sites of 45S rDNA. Chromosomal localization of the rDNA varied, which suggests that chromosome rearrangements took place during Anthoxanthum genome evolution.

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Can prolonged exposure to low VPD disturb the ABA signalling in stomatal guard cells?

The response of stomata to many environmental factors is well documented. Multiple signalling pathways for abscisic acid (ABA)-induced stomatal closure have been proposed over the last decades. However, it seems that exposure of a leaf for a long time (several days) to some environmental conditions generates a sort of memory in the guard cells that results in the loss of suitable responses of the stomata to closing stimuli, such as desiccation and ABA. In this review paper we discuss changes in the normal pattern of signal transduction that could account for disruption of guard cell signalling after long-term exposure to some environmental conditions, with special emphasis on long-term low vapour pressure deficit (VPD).

The Arabidopsis thaliana MYB60 promoter provides a tool for the spatio-temporal control of gene expression in stomatal guard cells

Plants have evolved different strategies to resist drought, of which the best understood is the abscisic acid (ABA)-induced closure of stomatal pores to reduce water loss by transpiration. The availability of useful promoters that allow for precise spatial and temporal control of gene expression in stomata is essential both for investigating stomatal regulation in model systems and for biotechnological applications in field crops. Previous work indicated that the regulatory region of the transcription factor AtMYB60 specifically drives gene expression in guard cells of Arabidopsis, although its activity is rapidly down-regulated by ABA. Here, the activity of the full-length and minimal AtMYB60 promoters is reported in rice (Oryza sativa), tobacco (Nicotiana tabacum), and tomato (Solanum lycopersicum), using a reporter gene approach. In rice, the activity of both promoters was completely abolished, whereas it was spatially restricted to guard cells in tobacco and tomato. To overcome the negative effect of ABA on the AtMYB60 promoter, a chimeric inducible system was developed, which combined the cellular specificity of the AtMYB60 minimal promoter with the positive responsiveness to dehydration and ABA of the rd29A promoter. Remarkably, the synthetic module specifically up-regulated gene expression in guard cells of Arabidopsis, tobacco, and tomato in response to dehydration or ABA. The comparative analysis of different native and synthetic regulatory modules derived from the AtMYB60 promoter offers new insights into the functional conservation of the cis-mechanisms that mediate gene expression in guard cells in distantly related dicotyledonous species and provides novel tools for modulating stomatal activity in plants.

Production of Amphidiploids of the Hybrids between Solanum macrocarpon and Eggplant

To restore fertility of the F1 between Solanum macrocarpon and eggplant, amphidiploids were produced through doubling of chromosomes by colchicine treatment. Shoot tips and axillary buds of F1 plants were kept for 2 and 4 days in liquid Murashige and Skoog (MS) media with 0.05% colchicine. As a result of colchicine treatment, two amphidiploids were identified by observing root tip cell chromosome number, stomatal guard cell size, and pollen characteristics. The amphidiploids contained 48 chromosomes, twice the normal diploid number of 24. Stomata size and pollen diameter were significantly larger in amphidiploids than F1. Flower diameter and length and width of anther, petal, and sepal were significantly larger in the amphidiploids than in F1. Pollen stainability was 40% in amphidiploids but only 0.86% in the diploid F1. The amphidiploids, after selfing and backcrossing with S. macrocarpon, set fruits with healthy seeds. Therefore, production of amphidiploids by colchicine treatment restored the pollen and seed fertility of F1 between S. macrocarpon and eggplant. The amphidiploids produced in this study would be very useful in future breeding programs of eggplant.

A Major Facilitator Superfamily Transporter Plays a Dual Role in Polar Auxin Transport and Drought Stress Tolerance inArabidopsis

Many key aspects of plant development are regulated by the polarized transport of the phytohormone auxin. Cellular auxin efflux, the rate-limiting step in this process, has been shown to rely on the coordinated action of PIN-formed (PIN) and B-type ATP binding cassette (ABCB) carriers. Here, we report that polar auxin transport in the Arabidopsis thaliana root also requires the action of a Major Facilitator Superfamily (MFS) transporter, Zinc-Induced Facilitator-Like 1 (ZIFL1). Sequencing, promoter-reporter, and fluorescent protein fusion experiments indicate that the full-length ZIFL1.1 protein and a truncated splice isoform, ZIFL1.3, localize to the tonoplast of root cells and the plasma membrane of leaf stomatal guard cells, respectively. Using reverse genetics, we show that the ZIFL1.1 transporter regulates various root auxin-related processes, while the ZIFL1.3 isoform mediates drought tolerance by regulating stomatal closure. Auxin transport and immunolocalization assays demonstrate that ZIFL1.1 indirectly modulates cellular auxin efflux during shootward auxin transport at the root tip, likely by regulating plasma membrane PIN2 abundance. Finally, heterologous expression in yeast revealed that ZIFL1.1 and ZIFL1.3 share H(+)-coupled K(+) transport activity. Thus, by determining the subcellular and tissue distribution of two isoforms, alternative splicing dictates a dual function for the ZIFL1 transporter. We propose that this MFS carrier regulates stomatal movements and polar auxin transport by modulating potassium and proton fluxes in Arabidopsis cells.

ATML1 promotes epidermal cell differentiation in Arabidopsis shoots

Molecular mechanisms that generate distinct tissue layers in plant shoots are not well understood. ATML1, an Arabidopsis homeobox gene, is expressed in the outermost cell layer, beginning at an early stage of development. The promoters of many epidermis-specific genes, including ATML1, contain an ATML1-binding site called an L1 box, suggesting that ATML1 regulates epidermal cell fate. Here, we show that overexpression of ATML1 was sufficient to activate the expression of epidermal genes and to induce epidermis-related traits such as the formation of stomatal guard cells and trichome-like cells in non-epidermal seedling tissues. Detailed observation of the division planes of these ectopic stomatal cells suggested that a near-surface position, as well as epidermal cell identity, were required for regular anticlinal cell division, as seen in wild-type epidermis. Moreover, analyses of a loss-of-function mutant and overexpressors implied that differentiation of epidermal cells was associated with repression of mesophyll cell fate. Collectively, our studies contribute new information about the molecular basis of cell fate determination in different layers of plant aerial organs.

High-resolution imaging andin situcutting of leaf epicuticular waxes of the biomass plantMiscanthus sinensis

The leaf surface features of the biomass plant Miscanthus sinensis were investigated by electron and ion beam microscopy. Fully developed leaves were collected from the plant, air-dried and examined by field emission scanning electron microscopy. Stomata and longitudinal stripes were present on both the adaxial and abaxial leaf surfaces. Longitudinally aggregated rodlets surrounded the stomata and formed hollow cylinders or chimney-like structures. With varying lengths up to ∼10 μm, the rodlets were curved (<1 μm in diameter) and almost occluded the stomata. As another type of epicuticular wax, platelets were observed in the vicinity of the stomata. The platelets were arranged into rather radially assembled clusters. As a novel approach for cross sectioning native epicuticular waxes, focused ion beam milling allowed precise in situ cutting of the stomata and epicuticular waxes. The longitudinally aggregated rodlets of the stomatal chimneys of M. sinensis were derived from the stomatal guard cells. These results suggest that the epicuticular waxes of M. sinensis can be categorized as stomatal chimneys (Strelitzia type) and rosettes (Fabales type). Combined electron and ion beam microscopy can help unravel the ultrastructure and vertical profile of epicuticular waxes in a range of plant taxa.

A Dof Transcription Factor, SCAP1, Is Essential for the Development of Functional Stomata in Arabidopsis

Stomata are highly specialized organs that consist of pairs of guard cells and regulate gas and water vapor exchange in plants [1-3]. Although early stages of guard cell differentiation have been described [4-10] and were interpreted in analogy to processes of cell type differentiation in animals [11], the downstream development of functional stomatal guard cells remains poorly understood. We have isolated an Arabidopsis mutant, stomatal carpenter 1 (scap1), that develops irregularly shaped guard cells and lacks the ability to control stomatal aperture, including CO2-induced stomatal closing and light-induced stomatal opening. SCAP1 was identified as a plant-specific Dof-type transcription factor expressed in maturing guard cells, but not in guard mother cells. SCAP1 regulates the expression of genes encoding key elements of stomatal functioning and morphogenesis, such as K(+) channel protein, MYB60 transcription factor, and pectin methylesterase. Consequently, ion homeostasis was disturbed in scap1 guard cells, and esterification of extracellular pectins was impaired so that the cell walls lining the pores did not mature normally. We conclude that SCAP1 regulates essential processes of stomatal guard cell maturation and functions as a key transcription factor regulating the final stages of guard cell differentiation.

Nitric oxide in guard cells as an important secondary messenger during stomatal closure.

The modulation of guard cell function is the basis of stomatal closure, essential for optimizing water use and CO2 uptake by leaves. Nitric oxide (NO) in guard cells plays a very important role as a secondary messenger during stomatal closure induced by effectors, including hormones. For example, exposure to abscisic acid (ABA) triggers a marked increase in NO of guard cells, well before stomatal closure. In guard cells of multiple species, like Arabidopsis, Vicia and pea, exposure to ABA or methyl jasmonate or even microbial elicitors (e.g., chitosan) induces production of NO as well as reactive oxygen species (ROS). The role of NO in stomatal closure has been confirmed by using NO donors (e.g., SNP) and NO scavengers (like cPTIO) and inhibitors of NOS (L-NAME) or NR (tungstate). Two enzymes: a L-NAME-sensitive, nitric oxide synthase (NOS)-like enzyme and a tungstate-sensitive nitrate reductase (NR), can mediate ABA-induced NO rise in guard cells. However, the existence of true NOS in plant tissues and its role in guard cell NO-production are still a matter of intense debate. Guard cell signal transduction leading to stomatal closure involves the participation of several components, besides NO, such as cytosolic pH, ROS, free Ca2+, and phospholipids. Use of fluorescent dyes has revealed that the rise in NO of guard cells occurs after the increase in cytoplasmic pH and ROS. The rise in NO causes an elevation in cytosolic free Ca2+ and promotes the efflux of cations as well as anions from guard cells. Stomatal guard cells have become a model system to study the signaling cascade mechanisms in plants, particularly with NO as a dominant component. The interrelationships and interactions of NO with cytosolic pH, ROS, and free Ca2+ are quite complex and need further detailed examination. While assessing critically the available literature, the present review projects possible areas of further work related to NO-action in stomatal guard cells.

Cell surface and cell outline imaging in plant tissues using the backscattered electron detector in a variable pressure scanning electron microscope.

BackgroundScanning electron microscopy (SEM) has been used for high-resolution imaging of plant cell surfaces for many decades. Most SEM imaging employs the secondary electron detector under high vacuum to provide pseudo-3D images of plant organs and especially of surface structures such as trichomes and stomatal guard cells; these samples generally have to be metal-coated to avoid charging artefacts. Variable pressure-SEM allows examination of uncoated tissues, and provides a flexible range of options for imaging, either with a secondary electron detector or backscattered electron detector. In one application, we used the backscattered electron detector under low vacuum conditions to collect images of uncoated barley leaf tissue followed by simple quantification of cell areas.ResultsHere, we outline methods for backscattered electron imaging of a variety of plant tissues with particular focus on collecting images for quantification of cell size and shape. We demonstrate the advantages of this technique over other methods to obtain high contrast cell outlines, and define a set of parameters for imaging Arabidopsis thaliana leaf epidermal cells together with a simple image analysis protocol. We also show how to vary parameters such as accelerating voltage and chamber pressure to optimise imaging in a range of other plant tissues.ConclusionsBackscattered electron imaging of uncoated plant tissue allows acquisition of images showing details of plant morphology together with images of high contrast cell outlines suitable for semi-automated image analysis. The method is easily adaptable to many types of tissue and suitable for any laboratory with standard SEM preparation equipment and a variable-pressure-SEM or tabletop SEM.

The effects of chromosome-doubling on selected morphological and phytochemical characteristics of Cichorium intybus L.

SummaryThe artificial induction of polyploidy is an efficient method by which to increase the production of secondary metabolites in various plant species, but it is often accompanied by changes in morphological and phytochemical characteristics. In this study, various concentrations of colchicine [0.00, 0.05, 0.10, 0.25, or 0.50% (w/v)] were applied to the seeds or shoot apical meristems of young seedlings of Cichorium intybus L. to induce tetraploidy. Microscopic studies were used to select putative polyploids, and flow cytometry analysis was conducted to confirm ploidy levels. Comparisons were made between selected structural and phytochemical characteristics of the parental diploid and induced tetraploid chicory plants. Based on the numbers of tetraploids obtained, treatment of shoot apical meristems was found to be more efficient than seed treatment. Structural characteristics such as the sizes of leaves, flowers, stomatal guard cells, pollen grains, and numbers of chloroplasts in paired guard cells were affected by an increase in ploidy level. These measurements also indicated that the induction of autotetraploidy in C. intybus L. had a significant effect on total phenolic compound and chlorogenic acid concentrations in leaves.

Photosynthesis of co-existing Phragmites haplotypes in their non-native range: are characteristics determined by adaptations derived from their native origin?

The Gulf Coast of North America (GC) is a ‘hot spot’ of Phragmites diversity as several lineages (defined according to the haplotypes of their chloroplast DNA) differing in origin, genetic traits and phenotype co-exist and interbreed in this area. We analysed differences in photosynthetic characteristics among and within four haplotypes to understand if differences in gas exchange can be attributed to adaptations acquired in their native ranges. We collected rhizomes of four GC haplotypes (I2, M1, M and AI; including the phenotypes ‘Land-type’, ‘Delta-type’, ‘EU-type’ and ‘Greeny-type’) and propagated them in a common controlled environment to compare photosynthesis–irradiance responses, CO2 responses, chlorophyll fluorescence, the activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), specific leaf area (SLA), pigment contents, stomatal density and guard cell length. The maximum light-saturated photosynthetic rate, Amax, in the haplotype I2 (Land-type) and haplotype M1 (Delta-type) (34.3–36.1 µmol CO2 m−2 s−1) was higher than that in the invasive Eurasian haplotype M (22.4 ± 2.3 µmol CO2 m−2 s−1). The Amax of haplotype AI (Greeny3-type) was 29.1 ± 4.0 µmol CO2 m−2 s−1 and did not differ from the Amax of the other haplotypes. The carboxylation rate (Vcmax) and electron transport rate (Jmax) followed the same pattern as Amax. The haplotypes also differed in SLA (17.0–24.3 m2 kg−1 dry mass) and pigment content, whereas stomatal density and guard cell length, Rubisco activity and chlorophyll fluorescence did not differ significantly among haplotypes. The high photosynthetic activity and gas-exchange capacity of the two haplotypes originating in tropical Africa and the Mediterranean area (haplotypes I2 and M1) are apparently adaptations derived from their native ranges. Hence, the haplotypes can be regarded as ecotypes. However, it remains unclear how these differences relate to plant competitiveness and fitness in the GC of North America environment.

Plant stem cell maintenance involves direct transcriptional repression of differentiation program

In animal systems, master regulatory transcription factors (TFs) mediate stem cell maintenance through a direct transcriptional repression of differentiation promoting TFs. Whether similar mechanisms operate in plants is not known. In plants, shoot apical meristems serve as reservoirs of stem cells that provide cells for all above ground organs. WUSCHEL, a homeodomain TF produced in cells of the niche, migrates into adjacent cells where it specifies stem cells. Through high-resolution genomic analysis, we show that WUSCHEL represses a large number of genes that are expressed in differentiating cells including a group of differentiation promoting TFs involved in leaf development. We show that WUS directly binds to the regulatory regions of differentiation promoting TFs; KANADI1, KANADI2, ASYMMETRICLEAVES2 and YABBY3 to repress their expression. Predictions from a computational model, supported by live imaging, reveal that WUS-mediated repression prevents premature differentiation of stem cell progenitors, being part of a minimal regulatory network for meristem maintenance. Our work shows that direct transcriptional repression of differentiation promoting TFs is an evolutionarily conserved logic for stem cell regulation.

Epicuticular Waxes and Stomata of Adult Scale Leaves of the Chinese Juniper Juniperus chinensis

Leaf surface structures were investigated in the Chinese juniper Juniperus chinensis by scanning electron microscopy. Adult scale leaves were collected from the tree, air-dried at room temperature, and sputter-coated with gold without further specimen preparation. Approximately fi ve stomata were locally distributed and arranged in clusters on the leaf surface. Stomata were ovoid and ca. 40 <TEX>${\mu}m$</TEX> long. The epicuticular wax structures of J. chinensis leaves were tubules and platelets. Numerous tubules were evident on the leaf regions where stomata were found. The tubules were cylindrical, straight, and ca. 1 <TEX>${\mu}m$</TEX> in length. They almost clothed the stomatal guard cells, and occluded the slit-shaped stomatal apertures. Moreover, the wax ridges were flat crystalloids that were connected to the surface by their narrow side. They did not have distinct edges, and their width/height ratio varied. In particular, the wax ridges could be discerned on the leaf regions where stomata were not present nearby. Since the wax ridges did not have distinct edges on their margin, they were identified as platelets. Instances were noted where platelets were oriented either parallel to each other or perpendicular to the cuticle surface. These results can be used in biomimetics to design the hierarchical structures for mimicking the plant innate properties such as hydrophobicity and self-cleaning effects of the leaf surface.

A novel indirect defence in Brassicaceae: Structure and function of extrafloral nectaries in Brassica juncea

While nectaries are commonly found in flowers, some plants also form extrafloral nectaries on stems or leaves. For the first time in the family Brassicaceae, here we report extrafloral nectaries in Brassica juncea. The extrafloral nectar (EFN) was secreted from previously amorphic sites on stems, flowering stalks and leaf axils from the onset of flowering until silique formation. Transverse sections at the point of nectar secretion revealed a pocket-like structure whose opening was surrounded by modified stomatal guard cells. The EFN droplets were viscous and up to 50% of the total weight was sugars, 97% of which was sucrose in the five varieties of B. juncea examined. Threonine, glutamine, arginine and glutamate were the most abundant amino acids. EFN droplets also contained glucosinolates, mainly gluconapin and sinigrin. Nectar secretion was increased when the plants were damaged by chewing above- and belowground herbivores and sap-sucking aphids. Parasitoids of each herbivore species were tested for their preference, of which three parasitoids preferred EFN and sucrose solutions over water. Moreover, the survival and fecundity of parasitoids were positively affected by feeding on EFN. We conclude that EFN production in B. juncea may contribute to the indirect defence of this plant species.

Variable Changes in Genome Size Associated with Different Polyploid Events in Plantago (Plantaginaceae)

The genus Plantago (Plantaginaceae) in New Zealand comprises species that can be difficult to delimit and contains a wide range of ploidy levels from diploid to 16-ploid. To investigate whether there have been changes in genome size associated with polyploid events and whether pollen and guard cell size can be used as an initial guide to identify polyploids, we have used flow cytometry with propidium iodide as the stain to measure the genome size (DNA C-value) in a range of Plantago species with different ploidy levels. Light microscopy was used to measure pollen diameter and guard cell length in these plants to see whether these could be used for ploidy level identification. Large differences in C-value were observed between species but the extent of changes associated with different polyploid events was highly variable. Pollen diameter was correlated with C-value but not necessarily with ploidy level. Guard cell lengths were different between diploids and polyploids but not between the different polyploid species. The significance of the changes in genome size and its relationship to pollen and stomatal guard cell size is discussed.

A comparison study on the interactions of two oligosaccharides with tobacco cells by time-resolved fluorometric method

The interactions of tobacco cells with two oceanic oligosaccharides, chitosan oligosaccharide (COS) and alginate derived oligosaccharide (AOS) were investigated and compared by time-resolved fluorometric method using two Eu3+ complexes as luminescence probes. The binding processes of two oligosaccharides and tobacco leaf cells (epidermal and stomatal guard cells) were characterized by the luminescence imaging method based on the reaction of tobacco cells with oligosaccharide (COS or AOS) conjugated to a highly fluorescent Eu3+ complex. In addition, the concentration changes of indole-3-acetic acid (IAA) and IAA-related peroxidase produced in the cells during the interaction of oligosaccharides (COS or AOS) and tobacco cells were also determined. The results indicate that the bioactivity of COS, with faster binding rate to the tobacco cells and stronger effect on the IAA generation in the cells, is remarkably higher than that of AOS. The comparison study on bioactivities of the two oligosaccharides reveals the relationship between structures and bioactivities of oligosaccharide, and suggests that COS should be more favorable to be used as a promotion reagent of plant growth in agriculture field.